Microcebus murinus: a novel promising non-human primate model of spinal cord injury

The number of people affected by spinal cord injuries (SCI) ranges from 2.5 to 4 million worldwide. Traumatic SCI induces a primary injury due to initial mechanical impact that causes focal cellular and blood-spinal cord barrier damages. Subsequently, secondary injuries resulting from in filtration of peripheral monocytes, ischemia, edema,in flammation, glial scar and cystic cavities formation as well as excitotoxicity trigger cellular demise and prevent spontaneous axonal regrowth. Secondary damages, including aggregation of extracellular matrix protein at the lesion epicenter, thus greatly amplify impairments induced by the initial mechanical damage.

Spinal cord injury across species:Many differences in neuroanatomical organization of the motor and sensory systems between rodents and primates may partially explain why responses to SCI vary considerably between species (Courtine et al., 2007). In particular,the corticospinal tract architecture in primates could explain their superior functional recovery after lateralized injuries compared to rodents (Friedli et al., 2015). The glial scar, resulting from SCI, is composed of activated astrocytes and microglia and forms a barrier to spontaneous axon regeneration. Temporal differences in glial response had been also reported between rodents and primates(Miller et al., 2012). In particular, in Macaca fascicularis, there is no substantial astrogliosis surrounding the lesion site (Wu et al., 2013).There is, however, an evident microglia/macrophage activation not only at the lesion site but also in adjacent spared tissues. Gene expression analysis in marmosets revealed a delayed glial scar formation and a prolonged in flammatory response compared to rodents,further supporting temporal differences in glial response after injury among species (Nishimura et al., 2014). Several non-human primate models have been developed to study the underlying mechanisms involved in SCI. Nevertheless, some limitations remain associated with these available non-human primate models of SCI. Macaca not only present a rather large body size that makes their handling more challenging but they also display a delayed sexual maturity, a low reproductive output and a long inter-birth interval (Fischer and Austad, 2011). Thus, smaller non-human primate model such as Callithrix Jacchus (marmosets) is also used in the SCI field (Okano et al., 2012). Iwanami and collaborators (Iwanami et al., 2005) have developed a model of graded contusive SCI at cervical level 5 in common marmoset. They assessed the motor function using grasping and cage-climbing tests as well as measurements of spontaneous motor activity. Magnetic resonance imaging follow up was done using a 1.5-Tesla apparatus and histological analyses were performed.However, no precise evaluation of glial cells response was reported.

Another small non-human primate, the grey mouse lemur (Microcebus murinus), presents many physiological and neuroanatomical similarities with human. In addition, their small size, rapid sexual maturity, high reproductive output and a short inter-birth interval makes them particularly useful for SCI investigations. However, the anatomical characterization of its spinal cord was rather limited. We thus first established a mini atlas to document the anatomical organization of the rachis and the spinal cord of Microcebus murinus using computerized tomography, ex vivo diffusion magnetic resonance imaging (MRI) and histological staining (Le Corre et al., 2017).

Microcebus murinus as a novel model of spinal cord injury Injury model:We then developed a new model of lateral spinal cord hemisection in Microcebus murinus (Figure 1A) and carried out detailed behavioral assessments (Le Corre et al., 2017). Following laminectomy of the posterior arch of the first lumbar vertebra, a lateral hemisection of the spinal cord was done under microscope using a micro-knife (Figure 1B). We also used longitudinal T2-weighted in vivo MRI to follow lesion evolution up to 3 months post-injury,followed by post-mortem ex-vivo high resolution T2-weighted-MRI(Figure 1C) and detailed histological assessments (Figure 1D).

Behavioral and imaging assessments: To evaluate post-operative de ficits, characterized by hind limb monoplegia ipsilateral to the lesion; we developed three different behavioral tests that were all performed 10, 5, and 2 days prior to surgery and then at 1, 3 and 7 days post-surgery. First, for open field test that consists of an evaluation of the spontaneous functional motor activity, lemurs were placed in an empty test arena and observed by two independent experimenters for at least 10 minutes and scored from 0 (no movement) to 6 (normal walking) . The open field test revealed severe motor deficits of the hind limb ipsilateral to the lesion up to 6 days post-lesion followed by a progressive recovery reaching over 80% of their initial scoring from 14 days onwards. The contralateral limb showed a transient impairment up to day 30 post-injury and then returned to normal. To evaluate fine motor function, we developed a grip test that consists of scoring the ability to grip a bar from 0 (no attempt to grip) to 2(normal grip). This test allowed identifying a gradual recovery of the hind limb ipsilateral to the lesion up to 70 days post-injury followed by a plateau at 75% of their initial scoring. The difference between the two hind limbs persisted up to the end of the experiment at 90 days post-injury. In parallel, we also performed automated gait analysis using CatWalk (Noldus, Wageningen, Netherlands) with many parameters analyzed related either to individual footprints (such as width and length of a complete paw print, pressure exerted by a paw,duration of contact of a paw with the glass plate, area of the print at max contact) or to the position of footprints in a step cycle (stride length, base of support, relative print position). CatWalk showed signi ficant alterations in motor function of the hind limb ipsilateral to the lesion up to 6 days after surgery, followed by a recovery period and a final return to normal from 14 days post-injury (Figure 1E, F).

In-vivo T2-weighted MRI (48 hours, 1, 4 and 12 weeks post-lesion)permitted to identify tissue re-organization at the lesion site over time resulting in an initially hypo-intense signal that was ultimately followed by a hyper-intense signal). Subsequent ex vivo T2-weighted MRI (Figure 1C) and histological assessments (Figure 1D) permitted to quantify the percentage of damage tissue at the epicenter. No difference between the three modalities (in & ex vivo MRI and histology) were observed with approximately 40% of damage area at the epicenter at 3 months post-injury.

Histological assessments: We next examine injury induced glial reactivity. We demonstrated a pronounced increase in glial fibrillary acidic protein (GFAP, 1:500; Dako, Glostrup, Denmark) expression at the lesion epicenter as well as adjacent to the lesion site (restricted to the grey matter rostral to the lesion). We also observed an increase in ionized calcium-binding adapter molecule 1 (IBA1, 1:200; Wako Pure Chemical Industries, Osaka, Japan) immunoreactivity adjacent to the lesion. These results thus revealed that astrocytic and microglial reactivity in Microcebus murinus persist 3 months following SCI. Interestingly we identi fied a pronounced increase in microglia/macrophage reactivity coinciding with a hyper-intense MRI signal within the ipsilateral dorsal funiculus rostral to the lesion.

Gaëtan Poulen, Florence Evelyne Perrin＊

University of Montpellier, Institut National de la Santé et de la

Recherche Médicale Unit 1198, Montpellier, France (Poulen G, Perrin FE)

Department of Neurosurgery, Gui de Chauliac Hospital, Montpellier

University Medical Center, Montpellier, France (Poulen G)

orcid:0000-0002-7630-0515 (Florence Evelyne Perrin)

Perspective: We have developed and characterized a novel model of SCI in a small non-human primates. We are now deepening our evaluation of the functional recovery of Microcebus murinus after SCI. In particular, behavioral assessments will be extended focusing on the grip test that can accurately discriminate fine motor impairments and detailed toe movements. In this regard, we are setting up a video monitoring analysis of Microcebus murinus along 3 axes while they are climbing on a scale consisting of bars of different diameters. To reliably quantify the precise force developed by the limbs, a bar with sensors detecting the force exerted by each hind limb may be used. This would allow an objective measurement of the force expressed in Newton as compared to a more subjective scoring based only on visual evaluation. A recent study has used a small iron bar mounted on a piezo-electric force to measure the pull strength of Microcebus murinus (Thomas et al., 2016). In this test, lemurs gripped a dowel with their hands and were then pulled away horizontally from the dowel. Peak forces in the horizontal direction were then recorded. An adaptation of this test could permit to also analyze the grip force of the hind limbs following SCI and thus to evaluate outcomes of a given therapeutic strategy. Another line of development is to evaluate sensory functions such as loss of sensation, enhanced abnormal sensation and neuropathic pain that is a common complication of SCI. An approach is to adapt the von-Frey filament test to Microcebus murinus following SCI. This test had been successfully used to evaluate responses to peripheral neuropathy following ligation of a spinal nerve in Macaca fascicularis (Palecek et al., 1992). Moreover, sensory changes addressed by von Frey test in non-human primate is currently one of the subject of studies developed by the California Spinal Cord Consortium. A simpler method, may be as for human to use different types of stimulation like light touch and pinprick to score responses of the primate (retraction or not of the stimulated hind limb).

In the model that we have recently developed, we used T2-weighted MRI (Multi Echo Multi Slices acquisition protocol) for in vivo and ex vivo acquisitions. However, to illustrate the mini atlas of Microcebus murinus spinal cord, we used ex vivo diffusion-weighted-MR axial images (dw-MRI) acquired using two sequential spinecho multi-slices protocols. Ex vivo diffusion MRI allows a better discrimination between the injured and intact tissue due to a higher signal-to-noise-ratio compared to ex vivo T2-weighetd images. The use of diffusion-weighted sequences should thus not only permit to better discriminate grey and white matters but also to measure more accurately the evolution of damage tissues over time. A future objective could be to correlate the percentage of lesion with the functional recovery rate.

Finally, from a mechanistic point of view, we will also analyze if a sub-population of astrocytes express markers of neuronal progenitors as we have recently identi fied in mice (Noristani et al., 2016).

Conclusion: As a conclusion, the new model that we have recently developed in Microcebus murinus can be used to promote translational research on SCI and represents an alternative to larger primates. Improvements in the follow-up after injury such as new tests measuring the fine motor movement and pull strength of the hind limbs after SCI, sensory function recovery and in vivo diffusion-weighted MRI sequences in this model will contribute and complement our understanding of SCI pathophysiology. Ultimately,it will be used for the development of therapeutic strategies to enhance functional regeneration following SCI.

This work was supported by the patient organizations “Verticale” and“Demain Debout Aquitaine”.

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Peer review:Externally peer reviewed.

Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under identical terms.

Open peer review reports:

Reviewer 1: Tania Cristina Leite de Sampaio e Spohr, Instituto Estadual do Cérebro Paulo Niemeyer, Brazil.

Comments to authors:The manuscript describes the use of the non-human primate model Microcebus murinus as a promising model of spinal cord injury. They also describe some results they have working with this model. The authors also describe shortly why the use of Macaca fascicularis and Callithrix jacchus has some limitations, as non-human primate model for SCI.

Reviewer 2:Carsten Theiss, Ruhr-Universitat Bochum, Germany.

Courtine G, Bunge MB, Fawcett JW, Grossman RG, Kaas JH, Lemon R,Maier I, Martin J, Nudo RJ, Ramon-Cueto A, Rouiller EM, Schnell L,Wannier T, Schwab ME, Edgerton VR (2007) Can experiments in nonhuman primates expedite the translation of treatments for spinal cord injury in humans? Nat Med 13:561-566.

Fischer KE, Austad SN (2011) The development of small primate models for aging research. ILAR J 52:78-88.

Friedli L, Rosenzweig ES, Barraud Q, Schubert M, Dominici N, Awai L,Nielson JL, Musienko P, Nout-Lomas Y, Zhong H, Zdunowski S, Roy RR, Strand SC, van den Brand R, Havton LA, Beattie MS, Bresnahan JC,Bezard E, Bloch J, Edgerton VR, et al. (2015) Pronounced species divergence in corticospinal tract reorganization and functional recovery after lateralized spinal cord injury favors primates. Sci Transl Med 7:302ra134.

Iwanami A, Yamane J, Katoh H, Nakamura M, Momoshima S, Ishii H,Tanioka Y, Tamaoki N, Nomura T, Toyama Y, Okano H (2005) Establishment of graded spinal cord injury model in a nonhuman primate: the common marmoset. J Neurosci Res 80:172-181.

Le Corre M, Noristani HN, Mestre-Frances N, Saint-Martin GP, Coillot C, Goze-Bac C, Lonjon N, Perrin FE (2017) A novel translational model of spinal cord injury in nonhuman primate. Neurotherapeutics doi:10.1007/s13311-017-0589-9.

Miller AD, Westmoreland SV, Evangelous NR, Graham A, Sledge J, Nesathurai S (2012) Acute traumatic spinal cord injury induces glial activation in the cynomolgus macaque (Macaca fascicularis). J Med Primatol 41:202-209.

Nishimura S, Sasaki T, Shimizu A, Yoshida K, Iwai H, Koya I, Kobayashi Y, Itakura G, Shibata S, Ebise H, Horiuchi K, Kudoh J, Toyama Y, Anderson AJ, Okano H, Nakamura M (2014) Global gene expression analysis following spinal cord injury in non-human primates. Exp Neurol 261:171-179.

Noristani HN, Sabourin JC, Boukhaddaoui H, Chan-Seng E, Gerber YN,Perrin FE (2016) Spinal cord injury induces astroglial conversion towards neuronal lineage. Mol Neurodegener 11:68.

Okano H, Hikishima K, Iriki A, Sasaki E (2012) The common marmoset as a novel animal model system for biomedical and neuroscience research applications. Semin Fetal Neonatal Med 17:336-340.

Palecek J, Dougherty PM, Kim SH, Palecková V, Lekan H, Chung JM, Carlton SM, Willis WD (1992) Responses of spinothalamic tract neurons to mechanical and thermal stimuli in an experimental model of peripheral neuropathy in primates. J Neurophysiol 68:1951-1966.

Thomas P, Pouydebat E, Brazidec ML, Aujard F, Herrel A (2016) Determinants of pull strength in captive grey mouse lemurs. J Zool 298:77-81.

Wu W, Wu W, Zou J, Shi F, Yang S, Liu Y, Lu P, Ma Z, Zhu H, Xu XM (2013)Axonal and glial responses to a mid-thoracic spinal cord hemisection in the Macaca fascicularis monkey. J Neurotrauma 30:826-839.